A longitudinal study on the impact of breastfeeding with or without formula milk on dental caries

Concerns exist about prolonged breastfeeding increasing dental caries risk, but evidence is mixed. This 2-year cohort study followed 486 toddlers, to examine the association between breastfeeding duration and caries at age 3. Caregivers reported feeding practices and potential confounders every 6 months. “Full breastfeeding” was defined as feeding breastmilk without formula milk regardless of other foods/liquids, whereas “any breastfeeding” was feeding breastmilk with/without formula milk. A calibrated dentist performed dental examinations. We used multivariable log-binomial and negative binomial regressions to estimate relative risks (RRs) and 95% confidence intervals (CIs) for caries prevalence and severity, adjusted for confounders. At 3-year-old, 60.3% of children exhibited caries (mean decayed-and-filled-teeth, dft: 3.3). Notably, full breastfeeding for 6–17 months reduced caries prevalence (RR = 0.84, 95%CI 0.73–0.98 for 6–11 months; RR = 0.78, 95%CI 0.63–0.96 for 12–17 months). Conversely, any breastfeeding ≥ 18 months significantly increased caries risk (RR = 1.45, 95%CI 1.31–1.60). Full breastfeeding ≥ 6 months or any breastfeeding 6–17 months was associated with lower dft scores in children. Our findings suggest a complex relationship between breastfeeding duration and caries. Full breastfeeding for moderate durations (6–17 months) offers protective benefits, while any breastfeeding ≥ 18 months increases risk in this population.

www.nature.com/scientificreports/The association between breastfeeding duration and caries has been examined in several studies; however, the results remain contradictory.While some studies reported that prolonged breastfeeding could be a risk factor for caries in primary dentition [7][8][9][10][11][12][13] , others showed that breastfeeding was either protective 14,15 or had no effect on early childhood caries [16][17][18] .Two systematic reviews and meta-analyses suggested that breastfeeding may protect against caries but breastfeeding beyond 12 months is associated with increased caries risk 19,20 .A recent systematic review, considering evidence from two cohort studies, indicated that breastfeeding up to 24 months does not increase caries risk, but a longer duration does 21 .These heterogenous findings justify the need for additional research, particularly cohort studies with careful control of relevant confounders.This prospective study aimed to investigate the association between breastfeeding duration and caries experience at 3 years of age.We hypothesized that breastfeeding for ≥ 12 months is a risk factor for caries in the primary dentition.

Study design and participants
The study protocol was conducted in accordance with the Declaration of Helsinki and approved by the institutional review boards of Khon Kaen University (HE592266) and University of Washington (HSD52258).Parents and/or caregivers provided written informed consent on behalf of the children for participation in the study.
This prospective cohort study was conducted in Muang District of Khon Kaen, in the northeast part of Thailand.Participants included all healthy one-year-old children (± 3 months) who attended local public health facilities for routine immunization between January and April 2017.Eighteen public health facilities were randomly selected based on geographical locations to cover all areas of the district.Of the 596 invited, 568 children were recruited after excluding those whose mothers planned to relocate.These children were followed up every 6 months for 2 years.The present analysis was based on children who remained in the cohort at three years old (n = 486) (Fig. 1).

Exposure and confounder assessment
Information on breastfeeding and other relevant factors was obtained through face-to-face interviews with mothers/caregivers by one interviewer using a structured questionnaire modified from our previous study 14 .Data on breastfeeding duration were collected at baseline and each follow-up visit through questions on breastmilk and other milk feeding habits, and age of weaning.This yielded a total of five surveys.Using the classification by Scott et al. 22 , breastfeeding was defined as: (1) full breastfeeding (providing an infant with breastmilk as the main source of nutrients, with or without water, other liquids, or foods, but not formula milk) and (2) any breastfeeding (providing an infant with breastmilk, with or without water, other liquids, foods, or formula milk).Additionally, children were classified according to their breastfeeding duration into four groups: < 6, 6-11, 12-17, and ≥ 18 months, following our previous work 14 .Full and any breastfeeding were classified separately.
Potential confounding factors were selected based on a review of the literature.Information collected at baseline include socio-demographic data of mothers/caregivers, frequency of teeth cleaning, fluoride toothpaste uses, and feeding habits (frequency of sleeping while milk feeding, frequency of water drinking after milk feeding, and number of meals per day).Information on sugar consumption between meals, including sugary drinks, sugary snacks, candies, jellies, desserts, and sweet fruits, was obtained at baseline and 3 years.Age of introduction to sugary foods was determined by interviewing mothers/caregivers at baseline and at each follow-up visit using the question, 'Did your child consume any sweets between meals during the day or at night last week?'Based on the responses collected across five surveys, we categorized the age of sugar introduction into three groups: < 12, 12-24, and > 24 months.The presence of visible plaque on buccal and lingual tooth surfaces was evaluated by a dentist at baseline and at 3 years, before caries assessment.

Outcome measurements
Dental caries was assessed when the children reached three years old.A calibrated dentist performed full-mouth examinations using modified WHO diagnostic criteria 23 .The numbers and locations of tooth surfaces with cavitated and non-cavitated (white spot) lesions were recorded separately.A child was considered to have caries if any cavitated carious lesions were present in at least one tooth.Before the caries assessment, all tooth surfaces were carefully wiped with dry gauze to remove plaque.Examinations were conducted under artificial light, using a mouth mirror, while the children were in a supine position.The dentist and caregiver sat knee to knee.After the examination, the caregivers received a report indicating whether further treatment was needed.Caregivers whose children had painful dental problems were instructed to take their children to see a dentist at a nearby hospital.

Statistical analysis
The baseline characteristics between children who completed the follow-up and those who dropped out were compared using Student's t-test for continuous variables and Pearson's Chi-square test for categorical variables.To compare participants' characteristics across the categories of breastfeeding duration, Pearson's Chi-square test or Fisher's exact test was used for categorical variables, and one-way ANOVA or Kruskal-Wallis test for continuous variables.To determine the association between breastfeeding duration and caries prevalence, log-binomial regression with a generalized linear model was performed to estimate relative risks (RRs) with 95% confidence intervals (Cis).Variables with a p value < 0.10 in the bivariate analysis or identified as potential confounding factors were considered for inclusion in the multivariable analyses.The change-in-estimate method 24 was employed to determine the inclusion of confounders.Confounders that changed the RR > 10% were included in the final model.Given that sugar consumption between meals was considered an important confounder, it was included in the final model, even though it changed the RR < 10%.To examine the association between breastfeeding duration and caries severity, the number of decayed and filled teeth (dft) was used as a count variable, and a negative binomial regression with a generalized linear model was employed to estimate RRs with 95% CIs.These models were fitted for overdispersion of caries incidence, assuming that the caries increment in this study had no excess zero.The analyses were performed using Stata version 10.1 (StataCorp, College Station, TX, USA).All statistical tests were two-sided, and a p value < 0.05 was considered as statistically significant.
The sample size was calculated with a ratio of 2 unexposed to 1 exposed, considering that 33.3% of Thai children continued breastfeeding after 12 months 25 .Based on a previous study 14 , caries prevalence among children aged 3-4 years who were breastfed for ≥ 12 months and < 12 months were 93% and 84.2%, respectively.A sample size of 477 was required to estimate the effect of breastfeeding for ≥ 12 months on dental caries, with 80% power and a 5% significance level.

Results
The flow of participants through the various stages of the study is shown in Fig. 1.Of the 568 eligible children, 486 (85.6%) were present at all visits and included in the analysis.There was no significant difference in all characteristics between children who remained in the study and those who dropped out (Table 1).The children were evenly distributed by sex, with an average age of 12.8 (standard deviation, SD = 1.1) months at baseline and 35.6 (SD = 1.4) months at the caries assessment visit.
Regarding breastfeeding, none of the children were exclusively breastfed for at least six months.More than half of the children were on full breastfeeding (55%) or any breastfeeding (57.8%) for at least 6 months.While any breastfeeding was continued in 18.1% of children for at least 18 months, full breastfeeding was continued in only 5.4% of children in the same duration (Table 1).
Mothers were the main caregivers of half of the children.Those raised by mothers or whose caregivers were younger, unemployed, and had education at the secondary school level or higher, tended to have a longer period of full or any breastfeeding (Table 2).Approximately two-thirds of the children who were breastfed for ≥ 18 months   www.nature.com/scientificreports/ reported sleeping during milk feeding more than three times a week.Children who were breastfed for a longer duration were more likely to have at least three meals per day and tended not to drink water after milk feeding.Of the 486 children, 293 (60.3%) had carious lesions at three years old.The prevalence and severity of caries were highest in children who were breastfed for less than 6 months.An inverse relationship between dental caries and breastfeeding duration was observed for full breastfeeding, i.e., dental caries decreased with a longer period of full breastfeeding.In contrast, the distribution of caries prevalence and severity by duration of any breastfeeding was U-shaped, with the shortest and longest duration groups being the most affected (Table 3 and  Table 4).Intra-rater reliability was measured by repeating the examination in 10% of the samples.Cohen's Kappa was 0.85, which was well above the expected minimum of 0.80.
Table 3 shows the association between breastfeeding duration and prevalence of dental caries.In the adjusted analysis, children who continued full breastfeeding for 6-17 months had a significantly lower risk of caries than those who received full breastfeeding for < 6 months (adjusted RR 0.84; 95% CI 0.73, 0.98 for 6-11 months and adjusted RR 0.78; 95% CI 0.63, 0.96 for 12-17 months).In contrast, any breastfeeding for ≥ 18 months was significantly associated with a 1.45 times increased risk of caries compared to any breastfeeding for < 6 months (adjusted RR 1.45; 95% CI 1.31, 1.60).
Regression analyses of the association between breastfeeding duration and caries severity are shown in Table 4. Compared with children who were fully breastfed for < 6 months, those who had a longer period of full breastfeeding had fewer teeth affected by caries.However, these differences were attenuated and became non-significant when adjusted for confounding factors.We observed a significantly lower dft score in children who continued any breastfeeding for 6-17 months compared to those who were breastfed for < 6 months (adjusted RR 0.76; 95% CI 0.59, 0.99 for 6-11 months and adjusted RR 0.70; 95% CI 0.50, 0.98 for 12-17 months).
Figure 2 illustrates the differences in the pattern of association between breastfeeding duration and caries experience according to full and any breastfeeding.Full breastfeeding duration was inversely associated with caries prevalence and dft in a linear fashion.In contrast, the association between any breastfeeding and dental caries was non-linear for both caries prevalence and dft.Table 3. Association between duration of breastfeeding and prevalence of dental caries at 3 years of age (n = 486).N, sample size; RR, relative risk; CI, confidence interval.† Adjusted for age and education of the main caregiver, frequency of drinking water after milk feeding at baseline, visible plaque at baseline, and sugar consumption between meals at 36 months.‡ Adjusted for education of main caregiver, age when other-milk feeding was started, frequency of sleeping while milk feeding at baseline, and sugar consumption between meals at 36 months.

Discussion
This prospective study, conducted in a non-fluoridated urban area in Thailand, examined the relationship between the duration of full breastfeeding, any breastfeeding and dental caries, separately.After adjusting for important confounders, we found that full breastfeeding for 6-17 months decreased the risk of dental caries in 3-year-old children compared with full breastfeeding for < 6 months.Similar trend was also observed for full breastfeeding for ≥ 18 months, albeit not statistically significant, due to limited sample size in this group.In contrast, caries prevalence was increased when any breastfeeding was continued for ≥ 18 months, compared to any breastfeeding for < 6 months.These findings suggest that caries risk associated with prolonged breastfeeding is not a result of breastfeeding per se but is likely due to the addition of formula milk.
Only a few studies have examined breastfeeding type in relation to caries.A study using the US nationally representative data of children aged 2-5 years did not find an association between dental caries and duration of breastfeeding, whether it was exclusive, full or overall 18 .The limitation of cross-sectional data was addressed with a potential for recall bias in the estimation of breastfeeding duration and introduction of other foods.The present findings confirmed the results of our previous birth cohort study in a rural/semi-urban district of Khon Kaen, which reported that full breastfeeding beyond 6 months was caries-protective 14 .The tendency that prolonged duration of any breastfeeding might increase the risk of caries was also observed, although the results were not statistically significant.Interestingly, our two cohort studies in non-fluoridated areas of Thailand found a non-linear association between the duration of any breastfeeding and caries experience, with < 6 months and ≥ 18 months groups being the most affected.These findings are consistent with a population-based cross-sectional survey in Australia that also observed a U-shaped association between breastfeeding duration and caries among children with no exposure to water fluoridation 7 .Non-linear J-shaped 10 and a trend of U-shaped associations 26 between breastfeeding duration and caries were also observed among young Japanese children in two non-fluoridated areas.These findings highlight the need to examine breastfeeding in intervals separating early (e.g., < 6 months) and late duration (≥ 18 months), rather than using specific binary cut-points 27 .
Our findings on any breastfeeding were in line with the results of previous systematic reviews.Tham and colleagues 20 suggested a protective effect of breastfeeding for up to 12 months against caries but identified a higher risk of dental caries in children breastfed beyond 12 months.Another meta-analysis by Cui and colleagues 19 reported that breastfeeding may protect against caries compared with never breastfeeding, while breastfeeding for ≥ 12 months was associated with an increased risk of dental caries compared to breastfeeding for < 12 months.Moynihan and colleagues also concluded that breastfeeding for ≤ 24 months does not increase caries risk, but a longer duration might elevate the risk 21 .A recent birth cohort study in the Netherlands confirmed the association between prolonged breastfeeding for > 12 months and an increased risk of childhood caries 13 .Nonetheless, another birth cohort study of Australian preschoolers (n = 965) did not observe an association between breastfeeding beyond one year and dental caries at 2-3 years of age.The authors addressed a limitation of the low prevalence of both dental caries and long duration of breastfeeding in this population, which could introduce a rare events bias 16 .A longitudinal study of Scottish children also failed to show the association of breastfeeding and dental caries at 1, 2, 3 and 4 years of age 17 .However, the study used 6 months as a cut-point for breastfeeding duration, which was not sensitive enough to detect the plausible effect of prolonged breastfeeding on caries 27 .
Mechanistically, there are several possible explanations for why prolonged full breastfeeding could decrease caries risk, while any breastfeeding beyond 18 months could increase the risk.The caries-protective effects may be derived from several glycoproteins in human breastmilk that could block the adhesion of Streptococcus mutans, a major cariogenic bacterial species, on tooth surfaces 28 .Moreover, the buffering capacity of breastmilk could help limit caries formation 29 and allow remineralization of non-cavitated lesions.However, human breastmilk contains high levels of lactose that might cause a pH drop below the critical level for enamel dissolution 30 .The addition of other carbohydrate sources, such as formula milk, may lead to a pH drop that exceeds the buffering capacity and enhances caries development 29 .As the levels of caries-protective elements in breastmilk tend to decline beyond 12 months 31 , the balance may be easier to be tipped with additional formula milk in prolonged duration of any breastfeeding over 18 months.
In a cross-sectional study involving 1-to 3-year-old children across 10 Latin American countries, Feldens and colleagues 32 found a relationship between breastfeeding duration and the age at which sugary products are introduced.The findings revealed that children who were breastfed for a shorter duration tended to be introduced to sugary products at an earlier age.However, in the current study, such a trend was not observed for either full or any breastfeeding (Table 2).This discrepancy might be explained by the high prevalence (97%) of children in our study receiving sugary foods before 12 months old.Other studies in Brazil found associations between early introduction to sugar 33 , increased exposure to cariogenic items before 12 months of age 34 , and the total daily feeding frequency at 12 months of age (including high-frequency breastfeeding and bottle-use) 35 with dental caries development.Therefore, it is crucial to take into account formula milk and other carbohydrates in the diets of children when studying the association between breastfeeding and dental caries.
A major strength of our study is its longitudinal cohort design.We collected breastfeeding information prospectively at multiple time points to ensure accurate breastfeeding status and minimize recall bias from caregiver reports.We obtained data from primary caregivers, who were closely involved in looking after the children, ensuring that the data closely reflected the actual information.Caries examinations were performed by a single dentist who was unaware of breastfeeding status and had excellent intra-examiner reliability.The retention rate after two years of follow-up was high (85.6%),and the characteristics of the participants who remained and those who were lost to follow-up were similar (Table 1), suggesting that selection bias was unlikely.Notably, we tried to minimize confounding by collecting rich information on potential confounders identified in the literature to control for in statistical analysis.Furthermore, we examined full and any breastfeeding separately and classified the duration into four categories.This approach enabled us to elucidate the complex associations between breastfeeding and caries for each duration.
This study had some limitations.Information on feeding practices was collected through interviews.There is a potential for recall bias and social desirability bias which may lead to exposure misclassification, even though we gathered breastfeeding information prospectively at several time points to minimize bias.Although we adjusted for important confounding factors, there could still be residual confounding from unmeasured factors, such as frequency of sleeping during feeding and age at introduction of complementary foods.Finally, the generalizability of our results may be limited to the population with similar patterns of breastfeeding and ECC rates.However, the biological mechanisms linking breastfeeding duration to caries should be the same across populations.
In conclusion, our findings support the value of continued breastfeeding throughout the first year of life and beyond, as recommended by leading organizations.A longer duration of full breastfeeding can protect against early childhood caries.However, any breastfeeding (with or without formula milk) for ≥ 18 months increases the caries prevalence.Therefore, breastfeeding practices should be strongly encouraged, along with urging caregivers to provide proper oral hygiene and dietary practices for children.Further prospective cohort studies with detailed categorization of breastfeeding type (i.e., full and any breastfeeding) and duration are needed to strengthen this evidence.

Figure 1 .
Figure 1.Flow chart of child participants through various stages of the study.All participants lost to follow-up were due to relocation to another district or province.
www.nature.com/scientificreports/n Duration of full breastfeeding in months (%) Duration of any breastfeeding in months (

Figure 2 .
Figure 2. Adjusted relative risks of dental caries (A) prevalence and (B) decayed and filled teeth (dft) at 3 years of age by 4 breastfeeding groups.

Table 1 .
Baseline characteristics of the participants included in the analysis and those who dropped out.Data presented as %, unless otherwise stated.N, sample size; SD, standard deviation; THB, Thai Baht (1 USD = 35.74THB).

Table 2 .
Baseline sociodemographic characteristics and potential confounders according to the duration of breastfeeding (n = 486).Data presented as % unless otherwise stated.N, sample size; SD, standard deviation.

Table 4 .
§ Pearson Chi-square test.Boldface indicates statistical significance.Association between duration of breastfeeding and dft score at 3 years of age (n = 486).N, sample size; dft, decayed and filled teeth; SD, standard deviation; RR, relative risk; CI, confidence interval.† Adjusted for education of the main caregiver, frequency of sleeping while milk feeding at baseline, frequency of cleaning teeth at baseline, and sugar consumption between meals at 36 months.‡ Adjusted for education of main caregiver, frequency of cleaning teeth at baseline, and sugar consumption between meals at 36 months.§ Kruskal Wallis test.Boldface indicates statistical significance.